Adsorbent for eliminating hepatitis C virus, adsorber, and adsorption method

ABSTRACT

An adsorbent for removing hepatitis C virus which has the ability to adsorb HCV particles, particularly immune-complex HCV particles, from a patient&#39;s body blood safely and with high efficiency and high selectivity for enhancing the efficacy of interferon therapy, an HCV adsorption apparatus including said adsorbent, and a adsorbing method for removing HCV are provided. 
     An adsorbent for removing hepatitis C virus which comprises a compound capable of adsorbing hepatitis C virus as immobilized on a water-insoluble carrier, an adsorption apparatus including said adsorbent, and an adsorbing method for removing HCV.

TECHNICAL FIELD

The present invention relates to an adsorbent for removing hepatitis Cvirus which is capable of selectively adsorbing hepatitis C virus frombody fluids such as blood, plasma, etc. to thereby expedite the cure forhepatitis C, an adsorption apparatus including said adsorbent, and anadsorbing method for removing hepatitis C virus.

PRIOR ART

With the successful cloning of the RNA virus genome of hepatitis C virusin 1989 (Q. L. Choo et al.: Science, 244, 359, 1989), it became possibleto assay anti-hepatitis C virus antibody using a recombinant protein. Asa result, most of the hepatitis termed non-A, non-B hepatitis in thepast were found to be hepatitis C. Thus, it is estimated that in Japantoday there are about 2,000,000 HCV carriers and, of them, 1,400,000have chronic hepatitis and 300,000 have cirrhosis (Shiro Iino: MedicalPractice in Gastroenterology-2, Hepatitis C, 11-17, 1993).

According to the Ministry of Health and welfare demographic statistics,the number of deaths due to primary liver cancer in 1992 was 27 thousand(1992 Demographic Statistics, Minister of Health and Welfare StatisticalInformation Bureau, Vol. 1, 1993) and approximately 70% of thecasualties were due to hepatocellular carcinoma associated withhepatitis C virus infection and it is by now considered that this cancerensues following the progression of chronic hepatitis to cirrhosis (S.Kaneko et al.: Intervirology, 37, 108, 1994; Eiki Matsushita et al.:Japanese Journal of Clinics, 53, 727, 1995 Special Issue). Therefore,hepatitis C can be said to be a refractory disease which progresses tocirrhosis to hepatocellular carcinoma.

The conventional therapy of hepatitis C is mostly built around restcure, dietary thereby, and pharmacotherapy using hepatoprotectantsand/or Chinese medicines. However, because the hepatitis virus cannot beremoved by such therapeutics, the cure rate is miserably low. This iswhy, in clinical practice, emphasis has been placed on the arrest ofprogression of chronic liver disease through palliation of local tissuenecrosis. Therefore, as the disease period is prolonged, many patientssuccumb to hepatocellular carcinoma, the serious outcome, throughcirrhosis as mentioned above.

Meanwhile, the mass production of interferons became feasible recentlyand those proteins were found to show not only antiviral activityagainst hepatitis C virus and its cognate RNA viruses in vitro (YasuyukiNinomiya et al.: The Clinical Report, 19, 231, 1985) but also protectiveactivity in mice infected with RNA viruses (M. Kramer et al.: J.Interferon Res., 3, 425, 1983). Accordingly, the utility of interferonsin clinical cases of hepatitis C has come into the focus of attention.

Actually, serum transaminase was normalized in some of the non-A, non-Bhepatitis cases which were treated with a recombinant interferon-alpha(J. H. Hoofnagel et al.: N. Eng. J. Med., 315, 1575, 1986) and in theadministration of an interferon to patients with hepatitis C, some casesbecame consistently negative to hepatitis C virus RNA in blood (K.Chayama et al.: Hepatology, 13, 1040, 1991; Hideki Ogiwara et al.:Japanese Journal of Gastroenterology, 88, 1420, 1991). In view of thoseresults, interferons have come to be broadly used in clinical practice.Thus, the therapy of hepatitis C has made a decisive step forward fromsymptomatic therapy to etiotropic therapy.

However, in the interferon therapies performed in about 200,000 cases oftype C chronic active hepatitis during the past several years in Japan,it was only in about 30% of cases that the virus could be eliminated andthe disease cured and in the remaining about 70% of cases the virussurvived and the therapy either proved ineffective or recurrences wereencountered (Migito Yano: Japanese Journal of Clinics, 53, 986, 1995Special Issue).

In the success or failure of a therapy, the hepatitis C virus gene type,the viral population in blood, and the stage of liver disease areimportant factors but, of all the factors involved, the viral populationin blood is the most important factor. For example, when the amount ofthe virus in 1 ml of the patient's blood was less than 1,000,000 copies,the virus could be eliminated from the body and the disease cured byadministration of an interferon in about 80% of cases but when theamount of the virus was over 1,000,000 copies, the cure rate was as lowas about 9% (Fumio Imazeki et al.: Japanese Journal of Clinics, 53,1017, 1995).

In addition to the above-mentioned amount of the virus, the inventors ofthe present invention found that the mode of existence of viralparticles in blood is also an important factor modifying the effect ofan interferon therapy. Thus, it has been reported that hepatitis C virusparticles in blood can be classified according to their suspensiondensity in blood into low-density particles with high infectivity andhigh-density particles with low infectivity. Therefore, the inventorsstudied the relationship of those viral particles varying in density tothe severity of illness and the interferon therapy and found thatwhereas the interferon therapy resulted in cure in 75% of patients withthe ratio of low-density viral particles to high-density viral particlesis 10:1, the cure rate in patients with the ratio of 1:10 was as low as13%.

It was also found that in blood, the low-density virus particles isbound to lipoprotein and the high-density virus particles toimmunoglobulin, thus existing as immune complexes (Akihito Sakai et al.:Japanese Journal of Gastroenterology, 92 (Special Issue), 1488, 1995).

It is, therefore, clear that the contemporary interferon therapy has thedrawback that the lower the blood viral population is or the lower theimmune-complex virus population is, the higher is the therapeuticresponse and conversely the higher the viral population is or the hitherthe immune-complex virus population is, the much lower is thetherapeutic response.

SUMMARY OF THE INVENTION

The present invention has for its object to provide an adsorbent forremoving hepatitis C virus which has the ability to adsorb hepatitis Cvirus particles, particularly immune-complex hepatitis C virusparticles, from a patient's body blood safely and with high efficiencyand high selectivity for enhancing the efficacy of interferon therapy,an hepatitis C virus adsorption apparatus including said adsorbent, andan adsorbing method for removing hepatitis C virus.

For accomplishing the above object, the inventors of the presentinvention made an intensive exploration for a compound which, whenimmobilized on a water-insoluble carrier and brought into contact with apatient's blood, should exhibit a high adsorbing affinity for hepatitisC virus but not for such proteins as albumin. As a result, the inventorsfound that an adsorbent fabricated by immobilizing a compound capable ofadsorbing hepatitis C virus, particularly a compound having a bindingaffinity for immunoglobulin and/or immune complex, on a water-insolublecarrier displays a remarkably high hepatitis C virus-adsorbingperformance. The present invention has been developed on the basis ofthe above finding.

The present invention, therefore, is directed to an adsorbent forremoving hepatitis C virus which comprises a compound capable ofadsorbing hepatitis C virus as immobilized on a water-insoluble carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of the relation between flowrate and pressure loss for glass columns packed with variouswater-insoluble carriers. The ordinate represents flow rate (cm/min.)and the abscissa represents pressure loss (kg/cm²);

FIG. 2 is a schematic cross-section view of the hepatitis C virusadsorption apparatus according to the invention; and

FIG. 3 is a diagram showing the pUCNTMK3P47 vector.

Each numeric symbol represents in the following.

1. outlet

2. inlet

3. absorbent for removing

4,5. means for preventing leakage of the absorbent

6. column

7. apparatus

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention is described in detail.

The adsorbent for removing hepatitis C virus according to the presentinvention comprises a water-insoluble carrier and, as immobilizedthereon, a compound capable of adsorbing hepatitis C virus.

The compound capable of adsorbing HCV is not particularly restrictedonly if it adsorbs hepatitis C virus but the preferred compound has abinding affinity for immunoglobulin and/or immune complex.

The preferred compound, among such compounds capable of adsorbinghepatitis C virus coupled to immunoglobulin and/or immune complex, is acompound which may preferentially and efficiently adsorb hepatitis Cvirus coupled to immunoglobulin and/or immune complex in comparison withhepatitis C virus particles as such.

More preferably, the above-mentioned compound having a binding affinityfor immunoglobulin and/or immune complex is an immunoglobulin-bindingprotein.

The immunoglobulin-binding protein includes but is not limited toprotein A, protein G, protein H, protein L, protein M, rheumatoidfactor, and complement.

Still more preferably, the compound having a binding affinity forimmunoglobulin and/or immunoglobulin complex is an anti-immunoglobulinantibody.

Even more preferably, the above-mentioned compound capable of adsorbinghepatitis C virus is a component of said immunoglobulin-binding proteinand/or anti-immunoglobulin antibody, which component is a fragmentprotein or a peptide containing a binding site for immunoglobulin and/orimmune complex or a derivative of said protein fragment or peptide.

Referring to the water-insoluble carrier, a porous carrier is apreferred example.

Preferably, the porous water-insoluble carrier has a mean pore diameterof from 10 to 1500 nm.

Another preferred form of the water-insoluble carrier is a substantiallynonporous carrier. Moreover, the water-insoluble carrier is preferablyhydrophilic.

The adsorbent for removing hepatitis C virus according to the presentinvention can be used for the purpose of removing hepatitis C virus frombody fluids inclusive of blood and plasma.

The adsorbent for removing hepatitis C virus can also be used for thepurpose of removing immune complex forms of hepatitis C virus from bodyfluids inclusive of blood and plasma.

The hepatitis C virus adsorption apparatus according to the presentinvention comprises a casing having an inlet and an outlet for admissionand discharge of a fluid and housing any of the above-mentionedadsorbents and a means for preventing leakage of said hepatitis C virusadsorbent from the casing.

The adsorbing method for removing hepatitis C virus comprises a step ofcontacting any of said adsorbents with a body fluid containing hepatitisC virus.

The HCV-containing body fluid for use in the method for removing HCVaccording to the present invention includes blood and plasma, amongother body fluids.

The preferred embodiments of the invention are now described, althoughthe invention is not limited to those specific embodiments.

The compound capable of adsorbing hepatitis C virus used in the presentinvention is a compound which is capable of adsorbing hepatitis C virusin a substantial measure and, as such, is not limited in kind.Preferably, however, it is a substance which may specifically bind theheavy chain or light chain of immunoglobulin and/or immunoglobulincomplex.

The above-mentioned substance capable of binding the heavy chain orlight chain of immunoglobulin and/or immune complex specificallyincludes the compounds which are generally called immunoglobulin-bindingproteins such as protein A, protein G, protein H, protein M, rheumatoidfactor, and complement, which can bind the Fc domain in the heavy chainof immunoglobulin G, and protein L which has a binding affinity for thelight chain (L. Bjorck: J. Immunol., 140, 1194, 1988; H. Gomi et al.: J.Immunol., 144, 4046, 1990; Hisayuki Doi: Meneki Rinsho (ClinicalImmunology), 23, 896, 1991) and anti-immunoglobulin antibodies.

There can also be mentioned those fragments of the above-mentionedsubstances which have a substantial binding affinity for immunoglobulinand/or immune complex, for example the peptides corresponding to the58-residue A through E domains, which are immunoglobulin-binding sites,of protein A (M. Uhlen et al.: J. Biol, Chem. 259, 1965, 1984), the FB29peptide which is a further abridgement of the B domain peptide ofprotein A (J. S. Huston et al.: Biophysical J., 62, 87, 1992), thepeptides corresponding to the 55-residue C1-C3 domains of protein G (B.Guss et al.: EMBO J., 5, 1567, 1986), the A domain peptide of protein H(H. Gomi et al.: ibid), the B1-B5 domain peptides of protein L (Bjorck,Laruth, et al., Japanese Kohyo Publication Hei-7-506573), and the CBP2peptide of complement C1q (M. A. Baumann et al.: J. Biol. Chem., 265,18414 (1990), etc., and other immunoglobulin-binding domain peptides ofso-called immunoglobulin-binding proteins, and their derivatives.

Furthermore, the Fab and F(ab) 2 fragments of rheumatoid factor oranti-immunoglobulin antibody, single-strand Fv polypeptide, etc. canalso be mentioned as representative examples.

The water-insoluble carrier which can be used in the present inventionis not particularly restricted but includes inorganic carriers such asglass beads, silica gel, etc., organic carriers such as syntheticpolymers, e.g. crosslinked polyvinyl alcohol, crosslinked polyacrylate,crosslinked polyacrylamide, crosslinked polystyrene, etc. andpolysaccharides such as crystalline cellulose, crosslinked cellulose,crosslinked agarose, crosslinked dextran, etc., and organic-organic ororganic-inorganic composite carriers consisting of such materials.

Particularly preferred are hydrophilic carriers, for such carriers arecharacterized in that the amount of non-specific adsorption isrelatively small and the adsorption selectivity to hepatitis C virus ishigh. The term “hydrophilic carrier” is used herein to mean a carrierthe constituent compound of which has a water-contact angle of notgreater than 60 degrees when it is molded in a flat sheet form.

The carrier of this kind is not particularly restricted but includescarriers made of polysaccharides such as cellulose, chitosan, Sepharose,dextran, etc., polyvinyl alcohol, saponified ethylene-vinyl acetatecopolymer, polyacrylamide, polyacrylic acid, polymethacrylic acid,poly(methyl methacrylate), polyacrylic acid-polyethylene alloy,polyacrylamide-polyethylene alloy, glass, and so forth.

Particularly, carriers containing OH groups are superior in adsorptivecapacity and selectivity. Moreover, porous cellulose gel has thefollowing advantageous features (1)-(4) and, as such, is one of the mostpreferred carriers for use as the water-insoluble carrier in thepractice of the invention.

(1) Because of its comparatively high mechanical strength and toughness,this gel is not easily disintegrated into dust-like fine particles bystirring, etc. and, when packed into a column, it is not appreciablycompacted or plugged even when a body fluid is passed through the bed ata high flow rate. Moreover, because of the porous structure, it is notliable to undergo dimensional change even when sterilized byautoclaving.

(2) Because it is made of cellulose, the gel is hydrophilic with a largenumber of hydroxyl groups available for binding to the ligand and is lowin nonspecific adsorption.

(3) Since a comparatively high strength can be retained even if the porevolume is increased, it may have an adsorptive capacity as large as asoft gel.

(4) Compared with synthetic polymer gel or other gels, this gel ishigher in safety.

The water-insoluble carriers mentioned above can be used each alone oras a suitable mixture of two or more species.

In consideration of its application as an adsorbent for removinghepatitis C virus and the mode of use, the water-insoluble carrier foruse in the present invention preferably has a large surface area and, inthis sense, is preferably a carrier having a large number of pores,namely porous.

The preferred mean pore diameter of said porous water-insoluble carrieris between 10 and 1500 nm. Hepatitis C virus particles are 50-55 nm indiameter and in order that such virus particles may be efficientlyadsorbed with a porous carrier, the pore size distribution profile ofthe carrier is preferably biased far toward the range larger than thediameter of virus particles. If the pore diameter is too large, thestrength of the carrier will be sacrificed and the surface areadecreased. The still more preferred mean particle diameter is between 50and 1250 nm.

On the other hand, the virus can be adsorbed even with a carrier whichis substantially nonporous. This kind of carrier can also be utilizedfor exploiting the advantage that the proteins and other componentsuseful for the body in the body fluid (blood, plasma, serum, etc.) arelittle adsorbed in the substantial absence of pores.

The term “substantially nonporous” is used in this specification toinclude porous carriers having very small pores (e.g. less than 10 nm).

Referring, further, to the porous structure of said water-insolublecarrier, it is preferable, in view of the adsorptive capacity per unitvolume of the adsorbent, that the pores should not be confined to thesurface but be distributed throughout the carrier and that the carrierhas a fractional pore volume of not less than 20% and a specific surfacearea of not less than 3 m²/g.

The form of said water-insoluble carrier is not particularly restrictedbut includes bead-form, fibrous form, and film form (inclusive of hollowfiber), among others. From the standpoint of hydrodynamics of the bodyfluid in extracorporeal circulation, a bead-form carrier is preferablyused. As to the mean particle diameter of said bead-form carrier, beadswithin the range of 10 and 2500 μm are easy to use. However, it ispreferable to use beads in the range of 25 and 800 μm.

The existence of functional groups available for immobilization of theligand on the surface of the water-insoluble carrier is beneficial toimmobilization.

The representative examples of such functional groups include hydroxyl,amino, aldehyde, carboxyl, thiol, silanol, amido, epoxy, succinylimido,acid anhydride, and other functional groups.

The above-mentioned water-insoluble carrier may be either a rigid geland a soft gel. For the application as an adsorbent for extracorporealcirculation therapies, it is important that, when packed in a column,there should not occur plugging, i.e. clogging of pores, on passage of abody fluid. Therefore, to insure a sufficient mechanical strength, thewater-insoluble carrier is preferably a rigid carrier.

In the context of the present invention, the rigid carrier is a carriersuch that when the gel, in the form of e.g. beads, is uniformly packedinto a glass cylindrical column (9 mm in. dia., 150 mm long) under thefollowing conditions and an aqueous fluid is passed through the packedcolumn, a linear relationship is obtained between pressure loss (ΔP) andflow rate up to 0.3 kg/cm².

For example, a glass cylindrical column (9 mm in. dia., 150 mm long)fitted with a 15 μm (pore diameter) filter at either end was uniformlypacked with an agarose gel (Bio-Rad, Biogel-A5m, particle diameter50-100 mesh), a vinyl polymer gel (Toyo Soda, Toyopearl HW-65, particlediameter 50-1000 μm), or a cellulose gel (Chisso Corporation,Cellulofine GC-700m, particle diameter 45-105 μm), water was introducedinto the column using a peristatic pump, and the relationship betweenflow rate and pressure loss (ΔP) was plotted (FIG. 1).

The flow rate (cm/min) was plotted on the ordinate and the pressure loss(kg/cm²) was plotted on the abscissa. In FIG. 1, ◯ represents ToyopearlHW-65, Δ represents Cellulofine GC-700m, and  represents Biogel-A5m.

As a result, whereas the flow rate increased in approximate proportionwith an increase in pressure in the case of Toyopearl HW-65 andCellulofine GC-700m, Biogel-A5m underwent compaction so that the flowrate could not be increased by raising the pressure.

In the immobilization of an immunoglobulin-binding protein or peptide onthe water-insoluble carrier in accordance with the present invention,the immobilization is preferably effected through a hydrophilic spacerin order to reduce the steric hindrance of the protein or peptide forimproving the adsorption efficiency and suppress non-specificadsorption.

The preferred hydrophilic spacer may for example be a polyalkylene oxidederivative available upon modification of the polyalkylene chain with asubstituent group such as carboxyl, amino, aldehyde, or epoxy at eitherend.

The compound having a binding affinity for immunoglobulin and/or immunecomplex which is to be immobilized on said water-insoluble carrier andthe organic compound as a spacer can be immobilized by any suitabletechnique. Among such techniques are those which are conventionally usedin the immobilization of proteins and peptides on carriers, such as themethods utilizing the epoxy reaction, Nic base reaction, condensationreaction using a carbodiimide reagent, active ester reaction, andcarrier crosslinking reaction using glutaraldehyde reagent.

In consideration of the fact that the adsorbent for removing hepatitis Cvirus according to the invention is an adsorbent chiefly used inextracorporeal circulation therapy and hemocatharsis, it is preferableto use an immobilization method which insures that in the sterilizationof the adsorbent and during such therapy, the proteins etc. will notreadily be released out from the water-insoluble carrier. For example,the following methods can be mentioned.

(1) The method which comprises reacting the carboxyl group of thecarrier with N-hydroxysuccinimide to substitute a succinimidoxycarbonylgroup and causing it to react with the amino group of the protein orpeptide (active ester method).

(2) The method which comprises subjecting the amino or carboxyl group ofthe carrier to condensation reaction with the carboxyl or amino group ofthe protein or peptide in the presence of a condensing agent such asdicyclohexylcarbodiimide (condensation method).

(3) The method in which the protein or peptide is crosslinked using acompound having two or more functional groups, such as glutaraldehyde(carrier crosslinking method).

For suppressing the release and elution of the protein from the carrier,immobilization is preferably effected by covalent bonding.

The adsorbing method for removing hepatitis C virus from a body fluid bycontacting a carrier carrying a compound capable of adsorbing hepatitisC virus with a body fluid such as blood, plasma or serum can bepracticed in a variety of manners. Specifically, for example, thefollowing methods can be mentioned.

(1) The method which comprises withdrawing a body fluid from thepatient's body, pooling it in a bag or the like, mixing the adsorbentwith the body fluid there to remove hepatitis C virus, filtering off theadsorbent and returning the substantially hepatitis C virus-free bodyfluid to the body.

(2) The method which comprises providing a casing having an inlet and anoutlet and fitted with a filter permeable to the body fluid but not tothe adsorbent across said outlet, packing the casing with the adsorbent,and passing the body fluid through the packed adsorbent.

Although both methods can be selectively employed, the second method (2)is expedient and simple procedure-wise. Moreover, when said casing orcolumn is built into a tubing system for extracorporeal circulation,hepatitis C virus can be efficiently and directly eliminated from thepatient's body fluid. The adsorbent for removing hepatitis C virusaccording to the invention is suited for the latter method.

The hepatitis C virus adsorption apparatus including the adsorbent forremoving hepatitis C virus according to the invention is now describedwith reference to the accompanying schematic drawing.

Referring to FIG. 2, the apparatus 7 has a liquid inlet or outlet 1, aliquid outlet or inlet 2, the hepatitis C virus adsorbent of theinvention 3, means 4 and 5 for preventing leakage of the adsorbent,through which the body fluid and its components may pass freely but theadsorbent cannot pass, and a column 6.

The geometry and material of the apparatus are not particularlyrestricted. However, it is preferable to use a cylindrical apparatushaving a capacity of about 20-400 mL and a diameter of about 2-10 cm.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples illustrate the present invention in furtherdetail without delimiting its metes and bounds. In this specification,various amino acid residues are indicated by the followingabbreviations.

Ala: L-alanine residue, Asp: L-aspartic acid residue, Asn: L-asparagineresidue, Cys: L-cysteine residue, Gln: L-glutamine residue, Glu:L-glutamic acid residue, Gly: L-glycine residue, Ile: L-isoleucineresidue, Leu: L-leucine residue, Lys: L-lysine residue, Phe:L-phenylalanine residue, Thr: L-threonine residue, Trp:L-tryptophanresidue, Tyr: L-tyrosine residue, Val: L-valine residue.

In this specification, the amino acid sequence of a peptide is describedin the conventional manner, assuming that its amino terminal(hereinafter referred to as N-terminus) is situated at the left end andits carboxyl terminal (referred to as C-terminus) at the right end.

EXAMPLE 1 Immobilization of an IgG-Binding Protein (Protein A) on aPorous Carrier (GCL 2000m)

Expoxy Activation of Cellulose Gel

The cellulosic porous rigid gel GCL-2000m (Chisso Corporation, globularprotein cutoff molecular weight 3×10⁶), 90 mL, was made up with water to180 mL, then 60 mL of 2 M sodium hydroxide was added, and the geltemperature was adjusted to 40° C. To this gel was added 21 mL ofepichlorohydrin, and the epoxidation reaction was carried out at 40° C.for 1 hour. After completion of the reaction, the gel was thoroughlyrinsed with water to provide an epoxy-activated cellulose gel.

Immobilization of Protein A

In 0.5 mL of 0.05 M borate buffer (pH 10.0) was dissolved 4 mg ofprotein A (Sigma), and 0.01 N NaOH/water was added so as to bring the pHto 10 and make a total volume of 1.0 mL (protein A solution). Thisprotein solution (total amount) was added to 1 mL of the aboveepoxy-activated cellulose gel and the mixture was shaken at 37° C. for16 hours and washed with a sufficient amount of PBS (10 mM phosphatebuffer supplemented with 150 mM sodium chloride) to provide GCL2000m-Protein A.

Quantitation of the Immobilized Protein

The protein A concentration was measured in the reaction mixture by HPLCbefore and after the immobilization reaction and the reaction rate wascalculated to find the amount of immobilization. It was found that 2.1mg of protein A was immobilized per mL of Protein A-GCL2000m.

EXAMPLE 2 Immobilization of an IgG-Binding Protein (Protein G) on aPorous Carrier (GCL2000m)

Using protein G (Pharmacia LKB) in lieu of protein A, the procedure ofExample 1 was otherwise repeated to provide GCL2000m-Protein G (3.2mg/mL).

EXAMPLE 3 Immobilization of the IgG-Binding Domain of Protein G on aPorous Carrier (Sepharose 6B)

Synthesis of a Peptide

A peptide having the amino acid sequence of 57 residues in the C3 domainof protein G with cysteine added to the N-terminus was synthesized bythe solid-phase method using Peptide Synthesizer Model 4170 (PharmaciaLKB).

Using 0.1 mmol of Fmoc-glutamine NovaSyn KA, a resin carrying theC-terminal glutamine, the deprotection reaction and condensationreaction were repeated in the direction toward the N-terminus forpeptide chain extension in accordance with the input program of theabove peptide synthesizer.

Thus, the cycle of removal of the α-amino-protecting group, i.e.9-fluorenylmethyloxycarbonyl (Fmoc), from the amino acid withpiperidine, washing with dimethylformamide (DMF), the condensationreaction using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate and diisopropylethylamine, and subsequent washing withDMF was repeated.

The amino acids were used in the forms of Fmoc-L-Ala, Fmoc-L-Asn(Trt),Fmoc-L-Asp(OtBu), Fmoc-L-Cys(Trt), Fmoc-L-Gln(Trt), Fmoc-L-Glu(OtBu),Fmoc-L-Gly, Fmoc-L-Ile, Fmoc-L-Leu, Fmoc-L-Lys(Boc), Fmoc-L-Phe,Fmoc-L-Thr(tBu), Fmoc-L-Trp, Fmoc-L-Tyr(tBu), and Fmoc-L-Val and each inan amount of about 5 molar equivalents (0.5 mmol) based on the substratein the vial. Here, Trt, OtBu, Boc, and tBu represent trityl, tert-butylester, tert-butyloxycarbonyl, and tert-butyl, respectively.

Deprotection and cleavage of the peptide chain

After completion of the reaction series for all the amino acids, thecarrier was washed successively with tert-amyl alcohol, acetic acid, anddiethyl ether on a 3G-3 pore glass filter and, then, dried in vacuo toprovide a dry carrier. To 1 g of the obtained carrier in the vial, 20 mLof trifluoroacetic acid (TFA), 260 μL of 1,2-ethanedithiol, and 780 μLof anisole were added and the mixture was stirred at room temperaturefor 1.5 hours.

Then, this mixture was filtered through a 3G-3 pore glass filter toremove the carrier and the filtrate was concentrated under reducedpressure at a temperature of 35° C. To the residue was added anhydrousdiethyl ether cooled ahead of time until a precipitate ceased to appearunder stirring, followed by centrifugation, and the crude peptide pelletwas recovered. This crude peptide was rinsed with several portions ofanhydrous diethyl ether and dried in vacuo to provide the objectivecrude peptide.

Purification of the Peptide

The above crude peptide was dissolved in 0.1% TFA and filtered through a0.2 μm membrane filter and the filtrate was subjected to highperformance liquid chromatography. For this HPLC, Model LC-10A System(Shimadzu) and, as the column, μ Bondasphere C18 (NipponMillipore-Waters) were used in a reversed phase. As the mobile phase,0.1% TFA/H₂O was used as solvent A and 0.1% TFA-80% (v/v)acetonitrile/H₂O for solvent B and elution was carried out on a lineargradient from Solvent A to solvent B.

The fraction corresponding to a chromatographic peak was collected.Fractional elution was repeated several times and the pooled fractionwas lyophilized to provide a purified peptide. This peptide wassubjected to amino acid analysis using Gas-phase Protein Sequencer 477(Applied Biosystems) and Hitachi Custom Ion Exchange Resin to confirmthat the peptide obtained had the amino acid sequence shown in SEQ IDNO:1.

Immobilization of the Peptide

An adsorbent was fabricated by immobilizing the above peptide on aporous Sepharose as follows. As the Sepharose, Thiopropyl-Sepharose 6B(Pharmacia LKB) was used. To 50 mg of Thiopropyl-Sepharose 6B was added50 ml of distilled water and the mixture was plated at room temperaturefor 15 minutes to let the resin swell. Then, distilled water was removedand replaced with 0.5 M NaCl-0.1 M Tris-HCl (pH 7.5) coupling buffer.

On the other hand, 4 mg of the above purified peptide was dissolved in400 μL of 0.5 M NaCl-0.1 M Tris-HCl (pH 7.5) coupling buffer. To thissolution was added 150 μL of the above swollen Thiopropyl-Sepharose 6B,and the mixture was stirred at 4° C. for 12 hours, whereby an adsorbentcarrying the purified peptide was obtained.

This peptide-carrying adsorbent was suction-filtered and the peptidecontent in the filtrate was determined by HPLC using the absolutecalibration curve method to find the peptide immobilization rate. Thispeptide-carrying adsorbent was washed well with 150 mM NaCl-10 mMphosphate buffer (pH 7.2) and suction-filtered to recover Sepharose6B-C3Ppt carrying 3.6 mg of the peptide per mL of the carrier.

EXAMPLE 4 Immobilization of an IgG-Binding Peptide (MK3P47) on a PorousCarrier (Kac)

Production of MK3P47 Peptide

A DNA coding for the MK3P47 peptide having the amino acid sequence shownin SEQ ID NO:2 was designed and synthesized so that it could be ligatedto pUCNT Vector (Japanese Kokai Publication Hei-4-212692) by utilizingits Nde I restriction enzyme site for the 5′-end and its Hind IIIrestriction enzyme site for the 3′-end. The nucleotide sequence of thesynthesized DNA is shown in SEQ ID NO:3.

The DNA having the above sequence was ligated to pUCNT Vector cleavedwith the restriction enzymes Nde I and Hind III (Takara Shuzo) inaccordance with the manual of Takara Shuzo's DNA Ligation Kit Ver. 2 toconstruct a pUCNTMK3P47 vector (FIG. 3).

Then, using the known technique, this pUCNTMK3P47 vector DNA wassubcloned in Escherichia coli HB101 (Irivitrogen) and a transformant wasselected with resistance to the antibiotic ampicillin as an indicator.

From this transformant, the plasmid DNA was extracted and sequenced bythe conventional procedure to confirm that it had a DNA sequenceconforming to the pUCNTMK3P47 vector design.

Then, this transformant was shake-cultured in 6L of L-Broth (5 g/L NaCl,10 g/L Bactotrypsin, 5 g/L yeast extract) at 37° C. for 20 hours and thecells were recovered by centrifugation (Hitachi RPR9-2 rotor, 4° C.,6000 rpm×20 min.).

The pellet obtained was suspended in 300 mL of TE Buffer (20 mMTris-HCl, 1 mM EDTA; pH 7.5), sonicated (BPANSON 250, in ice, 6 min.×3),and centrifuged (Hitachi RPR16 rotor, 4° C., 15000 rpm×20 min.) and thesupernatant was recovered.

The above supernatant was heat-treated at 70° C. for 10 minutes and thenrecentrifuged (Hitachi RPR16 rotor, 4° C., 15000 rpm×20 min.) to provide300 mL of a supernatant. From this supernatant, the objective MK3P47peptide was isolated as follows. Using a high performance liquidchromatograph (column: Waters' μBONDASPHERE 5μ C18 300A, 19.0×150 mm),40 ml of acetonitrile was passed at a flow rate of 5 ml/min to activatethe column and, then, 300 mL of the sample was passed at the same flowrate. The column was washed with 200 mL of 0.1% TFA+64% acetonitrile andthe objective MK3P47 peptide was then eluted with 200 mL of 0.1% TFA+40%acetonitrile.

This fraction was concentrated to 100 mL on an evaporator andlyophilized to provide 1.2 g of a high-purity sample of the peptide.

Expoxy Activation of Cellulose Gel

The prototype cellulosic porous rigid gel Kac with a globular proteincutoff molecular weight of >5×10⁶, prepared by the present applicant, 90mL, was made up with water to 180 mL. Then, 60 mL of 2 M sodiumhydroxide was added and the gel temperature was increased to 40° C. Tothis gel was added 21 mL of epichlorohydrin and the reaction wasconducted at 40° C. with stirring for 1 hour. After completion of thereaction, the gel was thoroughly rinsed with water to provide anepoxy-activated cellulose gel.

Immobilization of MK3P47 and determination of the amount of immobilizedprotein

Except that 50 mg of MK3P47 was used in lieu of 4 mg of protein A andepoxy-activated Kac was used in lieu of epoxy-activated GCL-2000m, theprocedure of Example 1 was otherwise repeated to provide Kac-MK3P47 (30mg/mL).

EXAMPLE 5 Immobilization of an IgG-Binding Peptide (MP47C) on AporousCarrier (Sephacryl S1000)

Peptide MP47C having the amino acid sequence shown in SEQ ID NO:4 wasprepared.

A DNA (coding for MP47C) of the nucleotide sequence shown in SEQ ID NO:5was designed and synthesized so that it could be ligated to pUCNT Vectoras in Example 4.

Thus, a pUCNTMP47C vector was prepared by ligating the above DNA topUCNT Vector by the same procedure as in Example 4.

Then, in the same manner as described in Example 4, an E. colitransformant was constructed and from 6L of its culture, 1.3 g of ahigh-purity sample of the objective peptide was obtained and used invarious studies.

Epoxy Activation of Sephacryl Gel

The cellulosic porous rigid gel (pore diameter 400 nm) Sephacryl S1000(Pharmacia LKB), 90 mL, was made up with water to 180 mL. Then, 60 mL of2M sodium hydroxide was added and the gel temperature was increased to40° C. To this gel was added 21 mL of epichlorohydrin and the reactionwas conducted at 40° C. with stirring for 1 hour. After completion ofthe reaction, the gel was thoroughly rinsed with water to provide anepoxy-activated Sephacryl gel.

Immobilization of MP47C and determination of the amount of immobilizedprotein

Except that 10 mg of MP47C was used in lieu of 4 mg of protein A and theepoxy-activated Sephacryl gel was used in lieu of epoxy-activatedGCL-2000m, the procedure of Example 1 was otherwise repeated to provideS1000-MP47C (7 mg/mL).

EXAMPLE 6 Immobilization of an IgG-Binding Peptide (MP47C) on aSubstantially Nonporous Carrier (Bac)

Epoxy Activation of Cellulose Gel

The prototype cellulosic rigid gel nonporous carrier (Bac) with aglobular protein cutoff molecular weight of <3×10⁴, 90 mL, was made upto 180 mL with water. Then, 60 mL of 2 M sodium hydroxide was added andthe gel temperature was increased to 40° C. To this gel was added 21 mLof epichlorohydrin, and the reaction was carried out at 40° C. withstirring for 1 hour. After completion of the reaction, the gel wasthoroughly rinsed with water to provide an epoxy-activated cellulosegel.

Immobilization of MP47C and determination of the amount of immobilizedprotein

Except that 30 mg of MP47C was used in lieu of 4 mg of protein A andepoxy-activated Bac in lieu of epoxy-activated GCL-2000m, theimmobilization procedure of Example 1 was otherwise repeated to provideBac-MP47C (20 mg/mL).

EXAMPLE 7 Immobilization of a Fragment (Fab) of the Anti-IgG Antibody ona Porous Carrier (CNBr-Activated Sepharose 6B)

CNBr-activated Sepharose 4B (Pharmacia LKB), 4 g, was swollen with asmall amount of 1 mM HCl/H₂O for 15 minutes and washed with 1 mM HCl/H₂Oand coupling buffer (pH 8.3, 0.5 M NaCl, 0.1 M NaHCO₃) in that order.

In 1 mL of coupling buffer was dissolved 1 mg of the Fab available onpapaine digestion (PIECE, ImmunoPure Fab Preparation Kit) of anti-humanIgG (Fab) antibody (Binding Site Co.). To this solution was added theabove washed gel and the reaction was carried out at 4° C. for 16 hours.

After the reaction mixture was washed with coupling buffer, block buffer(pH 8.3, 0.2 M glycine, 0.5 M NaCl, 0.1 M NaHCO₃) was added and reactedat room temperature for 2 hours.

The reaction product was washed with two kinds of after-treatmentbuffers (pH 4.0, 0.5 M NaCl, 0.1 M acetic acid-sodium acetate buffer andpH 8.0, 0.5 M NaCl, 0.1 M Tris-HCl buffer) alternately 3 times each toprovide Sepharose 4B-Anti-IgG Fab.

EXAMPLE 8 Immobilization of the Anti-IgG Antibody on a Porous Carrier(tresyl Toyopearl)

The anti-human IgG (Fc) antibody (Binding Site Co.), 1 mg, was dissolvedin 1 mL of coupling buffer (0.5 M NaCl, 0.1 M carbonate buffer) followedby addition of 200 mg of dry AF-tresyl-Toyopearl 650. The reaction wascarried out at 4° C. overnight.

After the reaction product was washed with 0.5 M NaCl/water, blockbuffer (pH 8.0, 0.5 M NaCl, 0.1 M Tris-HCl buffer) was added and reactedat room temperature for 2 hours.

This reaction mixture was further washed with 0.5 M NaCl/H₂O to provideToyopearl-anti-IgG.

EXAMPLE 9 Evaluation for the Hepatitis C Virus-Adsorbing Performance ofthe Synthesized Adsorbents

Adsorption Experiment

Each adsorbent, 100 μL, was taken in a vial, 100 μl of a patient serumcontaining hepatitis C virus was added, and the mixture was shaken at37° C. for 2 hours.

Determination of hepatitis C virus-adsorbing capacity

The above suspension was centrifuged at 5000 rpm for 1 minute and theamount of hepatitis C virus in the supernatant was determined as HCV RNA(Nippon Roche, Amplicore HCV Monitor).

As a control experiment, 100 μL of physiological saline in lieu of theadsorbent was taken in a vial and the above procedure was repeated todetermine the amount of hepatitis C virus in the solution.

The adsorption rate (%) of hepatitis C virus was calculated by means ofthe following equation.

Adsorption rate (%)=[(Vr−Vt)/Vr]×100

Vr:concentration of the virus in control solution

Vt:concentration of the virus in adsorption test supernatant

The results are shown in Table 1.

TABLE 1 HCV RNA HCV RNA Adsorp- adsorption test control ption Exam-supernatant solution rate ple Adsorbent (copy/mL) (copy/mL) (%) 1GCL2000m-Protein A 2.70E+04 7.90E+04 66 2 GCL2000m-Protein G 1.40E+047.90E+04 82 3 Sepharose6B-C3Ppt 8.10E+04 1.12E+05 28 4 Kac-MK3P473.20E+04 1.05E+05 70 5 S1000-MP47C 1.00E+04 1.05E+05 91 6 Bac-MP47C4.20E+04 1.05E+05 60 7 Sepharose4B- 4.80E+04 1.05E+05 54 AntiIgGFab 8Toyopearl-AntiIgG 3.60E+04 1.05E+05 66

EXAMPLE 10 Evaluation of the Synthesized Adsorbents

Adsorption Test

Each adsorbent, 100 μL, was taken in a vial, 100 μL of hepatitis Cvirus-containing patient serum was added, and the mixture was shaken at37° C. for 2 hours.

Determination of high-density HCV/low-density HCV

The HCV suspension obtained in each adsorption experiment and the HCVsuspension obtained using physiological saline in lieu of the adsorbentin otherwise the same manner were respectively admixed with anti-LDL andanti-IgG antibodies and the reaction was carried out at 4° C. for 16hours. The reaction mixture was centrifuged at 5000 rpm for 15 minutes,the pellet was recovered, and the amount of hepatitis C virus wasdetermined as HCV RNA (Nippon Roche, Amplicore HCV Monitor). Regardingthe HCV precipitated by anti-LDL antibody as low-density HCV and the HCVprecipitated by anti-IgG antibody as high-density HCV, the HCV RNA ratio(T/B=low-density HCV/high-density HCV) was calculated.

The results are shown in Table 2.

TABLE 2 T/B HCV T/B HCV adsorption control Example Adsorbent experimentexperiment 1 GCL2000m-Protein A 3/1 1/1  2 GCL2000m-Protein G 2/1 1/1  3Sepharose6B-C3Ppt 3/2 1/1  4 Kac-MK3P47 1/1 1/10 5 S1000-MP47C 1/1 1/106 Bac-MP47C 1/5 1/10 7 Sepharose4B- 1/3 1/10 AntiIgGFab 8Toyopearl-AntiIgG 1/3 1/10

INDUSTRIAL APPLICAPABILITY

As is clear from the following examples, the present invention providesa novel adsorbent having the ability to selectively adsorb and removethe hepatitis C virus present in body fluids and/or the ability toreduce the ratio of high-density HCV to low-density HCV. Furthermore, byusing a body fluid treating apparatus packed with the above adsorbent,hepatitis C virus can be selectively removed from body fluids such asblood, plasma, and serum.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 5 <210> SEQ ID NO 1 <211> LENGTH: 58<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: lgG - binding domain o #f protein G<400> SEQUENCE: 1 Cys Thr Thr Tyr Lys Leu Val Ile Asn Gly Ly#s Thr Leu Lys Gly Glu 1               5    #                10  #                15 Thr Thr Thr Lys Ala Val Asp Ala Ala Glu Th#r Ala Glu Lys Ala Phe             20       #            25      #            30 Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gl#y Val Trp Thr Tyr Asp         35           #        40          #        45 Asp Ala Thr Lys Thr Phe Thr Val Thr Glu     50              #    55 <210> SEQ ID NO 2 <211> LENGTH: 60 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: lgG - binding peptide <400> SEQUENCE: 2Met Lys Lys Lys Thr Thr Tyr Lys Leu Val Il #e Asn Gly Lys Thr Leu1               5    #                10   #                15Lys Gly Glu Thr Thr Thr Lys Ala Val Asp Al #a Glu Thr Ala Glu Lys            20       #            25       #            30Ala Phe Lys Gln Tyr Ala Asn Asp Asn Gly Va #l Asp Gly Val Trp Thr        35           #        40           #        45Tyr Asp Pro Ala Thr Lys Thr Phe Thr Val Th #r Glu     50              #     55              #    60 <210> SEQ ID NO 3 <211> LENGTH: 190<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: DNA coding for Sequence  #ID No: 2<400> SEQUENCE: 3catatgaaaa agaagaccac ctataaactg gttatcaacg gtaaaaccct ga#aaggtgaa     60accaccacca aggctgttga cgctgaaacc gctgaaaaag catttaaaca gt#atgctaac    120gacaacggtg tcgacggtgt ttggacctat gaccccgcta ccaaaacctt ta#ccgttacc    180 gaataagctt                 #                  #                   #       190 <210> SEQ ID NO 4 <211> LENGTH: 58<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: lgG - binding peptide <400> SEQUENCE: 4Met Thr Thr Tyr Lys Leu Val Ile Asn Gly Ly #s Thr Leu Lys Gly Glu 1              5    #                10   #                15Thr Thr Thr Lys Ala Val Asp Ala Glu Thr Al #a Glu Lys Ala Phe Lys            20       #             25      #            30Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Va #l Trp Thr Tyr Asp Pro        35           #        40           #        45Ala Thr Lys Thr Phe Thr Val Thr Glu Cys     50               #    55<210> SEQ ID NO 5 <211> LENGTH: 184 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: DNA coding for Sequence  #ID No. 4<400> SEQUENCE: 5catatgacca cctataaact ggttatcaac ggtaaaaccc tgaaaggtga aa#ccaccacc     60aaggctgttg acgctgaaac cgctgaaaaa gcatttaaac agtatgctaa cg#acaacggt    120gtcgacggtg tttggaccta tgaccccgct accaaaacct ttaccgttac cg#aatgctaa    180 gctt                  #                  #                   #            184

What is claimed is:
 1. An adsorbent for removing hepatitis C virus whichcomprises a compound capable of adsorbing hepatitis C virus asimmobilized on a water-insoluble carrier, wherein the compound capableof adsorbing hepatitis C virus is, a peptide having the amino acidsequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:4.
 2. The adsorbentfor removing hepatitis C virus according to claim 1 wherein thewater-insoluble carrier is a porous carrier.
 3. The adsorbent forremoving hepatitis C virus according to claim 2, wherein the porouscarrier has a mean pore diameter ranging from 10 to 1500 nm.
 4. Theadsorbent of claim 3 wherein the carrier has a fractional pore volume ofnot less than 20% and a specific surface area of not less than 3m²/g. 5.The adsorbent for removing hepatitis C virus according to claim 1,wherein the water-insoluble carrier is a substantially nonporouscarrier.
 6. The adsorbent for removing hepatitis C virus according toclaim 1 wherein the water-insoluble carrier is a hydrophilic carrier. 7.An apparatus for adsorbing hepatitis C virus which comprises a casinghaving an inlet and an outlet for admission and discharge of a fluid andhousing the adsorbent for removing hepatitis C virus according to claim1, and a means for preventing leakage of said adsorbent for removinghepatitis C virus from the casing.
 8. The adsorbent for removinghepatitis C virus according to claim 1 wherein the compound capable ofadsorbing hepatitis C virus is a peptide having the amino acid sequenceof SEQ ID NO:1.
 9. The adsorbent for removing hepatitis C virusaccording to claim 1 wherein the compound capable of adsorbing hepatitisC virus is a peptide having the amino acid sequence of SEQ ID NO:2. 10.The adsorbent for removing hepatitis C virus according to claim 1wherein the compound capable of adsorbing hepatitis C virus is a peptidehaving the amino acid sequence of SEQ ID NO:4.
 11. An adsorbent forremoving hepatitis C virus which comprises a compound capable ofadsorbing hepatitis C virus bound to immunoglobulin and/or immunecomplex as immobilized on a water-insoluble carrier, wherein saidcompound capable of adsorbing hepatitis C virus is bound toimmunoglobulin and/or immune complex is a peptide having the amino acidsequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:4.
 12. The adsorbentfor removing hepatitis C virus according to claim 11 wherein thewater-insoluble carrier is a porous carrier.
 13. The adsorbent forremoving hepatitis C virus according to claim 12 wherein the porouscarrier has a mean pore diameter ranging from 10 to 1500 nm.
 14. Theadsorbent for removing hepatitis C virus according to claim 13 whereinthe carrier has a fractional pore volume of not less than 20% and aspecific surface area of not less than 3 m²/g.
 15. The adsorbent forremoving hepatitis C virus according to claim 11 wherein thewater-insoluble carrier is a substantially nonporous earner.
 16. Theadsorbent for removing hepatitis C virus according to claim 11 whereinthe water-insoluble carrier is a hydrophilic carrier.
 17. An apparatusfor adsorbing hepatitis C virus which comprises a casing having an inletand an outlet for admission and discharge of a fluid and housing theadsorbent for removing hepatitis C virus according to claim 11, and ameans for preventing leakage of said adsorbent for removing hepatitis Cvirus from the casing.
 18. The adsorbent for removing hepatitis C virusaccording to claim 11 wherein the compound capable of adsorbinghepatitis C virus bound to immunoglobulin and/or immune complex is apeptide having the amino acid sequence of SEQ ID NO:1.
 19. The adsorbentfor removing hepatitis C virus according to claim 11 wherein thecompound capable of adsorbing hepatitis C virus bound to immunoglobulinand/or immune complex is a peptide having the amino acid sequence of SEQID NO:2.
 20. The adsorbent for removing hepatitis C virus according toclaim 11 wherein the compound capable of adsorbing hepatitis C virusbound to immunoglobulin and/or immune complex is a peptide having theamino acid sequence of SEQ ID NO:4.